Nucleotide Sequences Which Code for the tal Gene

ABSTRACT

The invention relates to an isolated polynucleotide from coryneform bacteria, comprising a polynucleotide sequence chosen from the group consisting of
     a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for a polypeptide which comprises the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,   b) polynucleotide which codes for a polypeptide which comprises an amino acid sequence which is identical to the extent of at least 70% to the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4   c) polynucleotide which is complementary to the polynucleotides of a) or b) and   d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c)
 
and a process for the preparation of L-amino acids, which comprises carrying out the following steps:
   a) fermentation of the desired L-amino acid-producing bacteria in which at least the tal gene is amplified,   b) concentration of the desired product in the medium or in the cells of the bacteria and   c) isolation of the L-amino acid.

The invention provides nucleotide sequences which code for the tal geneand a process for the fermentative preparation of amino acids, inparticular L-lysine, L-threonine, L-isoleucine and L-tryptophan, usingcoryneform bacteria in which the tal gene is amplified.

PRIOR ART

Amino acids, in particular L-lysine, are used in human medicine and inthe pharmaceuticals industry, but in particular in animal nutrition.

It is known that amino acids are prepared by fermentation by strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Becauseof their great importance, work is constantly being undertaken toimprove the preparation processes. Improvements to the processes canrelate to fermentation measures, such as e.g. stirring and supply ofoxygen, or the composition of the nutrient media, such as e.g. the sugarconcentration during the fermentation, or the working up to the productform by e.g. ion exchange chromatography, or the intrinsic outputproperties of the microorganism itself.

Methods of mutagenesis, selection and mutant selection are used toimprove the output properties of these microorganisms. Strains which areresistant to antimetabolites, such as e.g. the lysine analogueS-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites ofregulatory importance and produce L-amino acids, such as e.g. L-lysine,are obtained in this manner.

Methods of the recombinant DNA technique have also been employed forsome years for improving the strain of Corynebacterium strains whichproduce amino acids, by amplifying individual amino acid biosynthesisgenes and investigating the effect on the amino acid production.

Review articles in this context are to be found, inter alia, inKinoshita (“Glutamic Acid Bacteria”, in: Biology of IndustrialMicroorganism's, Demain and Solomon (Eds.), Benjamin Cummings, London,UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (AminoAcids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews inBiotechnology 15, 73-103 (1995)) and Sahm et al. (Annuals of the NewYork Academy of Science 782, 25-39 (1996)).

The importance of the pentose phosphate cycle for the biosynthesis andproduction of amino acids, in particular L-lysine, by coryneformbacteria is the subject of numerous efforts among experts.

Thus Oishi and Aida (Agricultural and Biological Chemistry 29, 83-89(1965)) report on the “hexose monophosphate shunt” of Brevibacteriumammoniagenes. Sugimoto and Shio (Agricultural and Biological [sic]Chemistry 51, 101-108 (1987)) report on the regulation of glucose6-phosphate dehydrogenase in Brevibacterium flavum.

OBJECT OF THE INVENTION

The inventors had the object of providing new measures for improvedfermentative preparation of amino acids, in particular L-lysine,L-threonine, L-isoleucine and L-tryptophan.

DESCRIPTION OF THE INVENTION

Amino acids, in particular L-lysine, are used in human medicine, in thepharmaceuticals industry and in particular in animal nutrition. There istherefore a general interest in providing new improved processes for thepreparation of amino acids, in particular L-lysine.

When L-lysine or lysine are mentioned in the following, not only thebase but also the salts, such as e.g. lysine monohydrochloride or lysinesulfate, are also meant by this.

The invention provides an isolated polynucleotide from coryneformbacteria, comprising a polynucleotide sequence chosen from the groupconsisting of

-   a) polynucleotide which is identical to the extent of at least 70%    to a polynucleotide which codes for a polypeptide which comprises    the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,-   b) polynucleotide which codes for a polypeptide which comprises an    amino acid sequence which is identical to the extent of at least 70%    to the amino acid sequences of SEQ ID NO. 2 or SEQ ID NO. 4,-   c) polynucleotide which is complementary to the polynucleotides    of a) or b) and-   d) polynucleotide comprising at least 15 successive nucleotides of    the polynucleotide sequence of a), b) or c).

The invention also provides the polynucleotide as claimed in claim 1,this preferably being a DNA which is capable of replication, comprising:

-   (i) a nucleotide sequence chosen from the group consisting of SEQ ID    NO. 1 and SEQ ID NO. 3 or-   (ii) at least one sequence which corresponds to sequence (i) within    the range of the degeneration of the genetic code, or-   (iii) at least one sequence which hybridizes with the sequence    complementary to sequence (i) or (ii), and optionally-   (iv) sense mutations of neutral function in (i).

The invention also provides

-   a polynucleotide as claimed in claim 4, comprising one of the    nucleotide sequences as shown in SEQ ID NO. 1 and SEQ ID NO. 3,-   a polynucleotide as claimed in claim 5, which codes for a    polypeptide which comprises the amino acid sequence as shown in SEQ    ID NO. 2 and SEQ ID NO. 4,-   a vector containing the polynucleotide as claimed in claim 1,-   and coryneform bacteria, serving as the host cell, which contain the    vector.

The invention also provides polynucleotides which substantially comprisea polynucleotide sequence, which are [sic] obtainable by screening bymeans of hybridization of a corresponding gene library, which comprisesthe complete gene with the polynucleotide sequence corresponding to SEQID NO. 1 or SEQ ID NO. 3, with a probe which comprises the sequence ofthe polynucleotide mentioned, according to SEQ ID NO. 1 or SEQ ID NO. 3or a fragment thereof, and isolation of the DNA sequence mentioned.

Polynucleotide sequences according to the invention are suitable ashybridization probes for RNA, cDNA and DNA, in order to isolate, in thefull length, cDNA which code for transaldolase and to isolate those cDNAor genes which have a high similarity of sequence with that of thetransaldolase gene.

Polynucleotide sequences according to the invention are furthermoresuitable as primers for the preparation of DNA of genes which code fortransaldolase by the polymerase chain reaction (PCR).

Such oligonucleotides which serve as probes or primers comprise at least30, preferably at least 20, especially preferably at least 15 successivenucleotides. Oligonucleotides which have a length of at least 40 or 50nucleotides are also suitable.

“Isolated” means separated out of its natural environment.

“Polynucleotide” in general relates to polyribonucleotides andpolydeoxyribonucleotides, it being possible for these to be non-modifiedRNA or DNA or modified RNA or DNA.

“Polypeptides” is understood as meaning peptides or proteins whichcomprise two or more amino acids bonded via peptide bonds.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID NO. 2 or SEQ ID NO. 4, in particular those with thebiological activity of transaldolase, and also those which are identicalto the extent of at least 70% to the polypeptide according to SEQ ID NO.2 or SEQ ID NO. 4, and preferably are identical to the extent of atleast 80% and in particular to the extent of at least 90% to 95% to thepolypeptide according to SEQ ID NO. 2 or SEQ ID NO. 4, and have theactivity mentioned.

The invention also provides a process for the fermentative preparationof amino acids, in particular L-lysine, L-threonine, L-isoleucine andL-tryptophan, using coryneform bacteria which in particular alreadyproduce an amino acid, and in which the nucleotide sequences which codefor the tal gene are amplified, in particular over-expressed.

The term “amplification” in this connection describes the increase inthe intracellular activity of one or more enzymes in a microorganismwhich are coded by the corresponding DNA, for example by increasing thenumber of copies of the gene or genes, using a potent promoter or usinga gene which codes for a corresponding enzyme having a high activity,and optionally combining these measures.

The microorganisms which the present invention provides can prepareL-amino acids, in particular L-lysine, from glucose, sucrose, lactose,fructose, maltose, Molasses, starch, cellulose or from glycerol andethanol. They can be representatives of coryneform bacteria, inparticular of the genus Corynebacterium. Of the genus Corynebacterium,there may be mentioned in particular the species Corynebacteriumglutamicum, which is known among experts for its ability to produceL-amino acids.

Suitable strains of the genus Corynebacterium, in particular of thespecies Corynebacterium glutamicum, are, for example, the knownwild-type strains

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Corynebacterium melassecola ATCC17965    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        and L-lysine-producing mutants or strains prepared therefrom,        such as, for example    -   Corynebacterium glutamicum FERM-P 1709    -   Brevibacterium flavum FERM-P 1708    -   Brevibacterium lactofermentum FERM-P 1712    -   Corynebacterium glutamicum FERM-P 6463    -   Corynebacterium glutamicum FERM-P 6464 and    -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium glutamicum DM58-1    -   Corynebacterium glutamicum DSM12866.        and L-threonine-producing mutants or strains prepared therefrom,        such as, for example    -   Corynebacterium glutamicum ATCC21649    -   Brevibacterium flavum BB69    -   Brevibacterium flavum DSM5399    -   Brevibacterium lactofermentum FERM-BP 269    -   Brevibacterium lactofermentum TBB-10        and L-isoleucine-producing mutants or strains prepared        therefrom, such as, for example    -   Corynebacterium glutamicum ATCC 14309    -   Corynebacterium glutamicum ATCC 14310    -   Corynebacterium glutamicum ATCC 14311    -   Corynebacterium glutamicum ATCC 15168    -   Corynebacterium ammoniagenes ATCC 6871        and L-tryptophan-producing mutants or strains prepared        therefrom, such as, for example    -   Corynebacterium glutamicum ATCC21850 and    -   Corynebacterium glutamicum KY9218 (pKW9901)

The inventors have succeeded in isolating the new tal gene of C.glutamicum which codes for transaldolase (EC 2.2.1.2).

To isolate the tal gene or also other genes of C. glutamicum, a genelibrary of this microorganism is first set up in E. coli. The setting upof gene libraries is described in generally known textbooks andhandbooks. The textbook by Winnacker: Gene and Klone, Eine Einführung indie Gentechnologie [Genes and Clones, An Introduction to GeneticEngineering] (Verlag Chemie, Weinheim, Germany, 1990) or the handbook bySambrook et al.: Molecular Cloning, A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1989) may be mentioned as an example. Awell-known gene library is that of the E. coli K-12 strain W3110 set upin λ vectors by Kohara et al. (Cell 50, 495-508 (1987)). Bathe et al.(Molecular and General Genetics, 252:255-265, 1996) describe a genelibrary of C. glutamicum ATCC13032, which was set up with the aid of thecosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the NationalAcademy of Sciences USA, 84:2160-2164) in the E. coli K-12 strain NM554(Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Börmann etal. (Molecular Microbiology 6(3), 317-326)) (1992)) in turn describe agene library of. C. glutamicum ATCC13032 using the cosmid pHC79 (Hohnand Collins, Gene 11, 291-298 (1980)). O'Donohue (The Cloning andMolecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genesfrom Corynebacterium glutamicum. Ph.D. Thesis, National University ofIreland, Galway, 1997) describes the cloning of C. glutamicum genesusing the λ Zap expression system described by Short et al. (NucleicAcids Research, 16: 7583). To prepare a gene library of C. glutamicum inE. coli it is also possible to use plasmids such as pBR322 (Bolivar,Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene,19:259-268). Suitable hosts are, in particular, those E. coli strainswhich are restriction- and recombination-defective. An example of theseis the strain DH5αmcr, which has been described by Grant et al.(Proceedings of the National Academy of Sciences USA, 87 (1990)4645-4649). The long DNA fragments cloned with the aid of cosmids canthen in turn be subcloned and subsequently sequenced in the usualvectors which are suitable for sequencing, such as is described e.g. bySanger et al. (Proceedings of the National Academy of Sciences of theUnited States of America, 74:5463-5467, 1977).

The DNA sequences obtained can then be investigated with knownalgorithms or sequence analysis programs, such as e.g. that of Staden(Nucleic Acids Research 14, 217-232 (1986)), the GCG program of Butler(Methods of Biochemical Analysis 39, 74-97 (1998)) the FASTA algorithmof Pearson and Lipman (Proceedings of the National Academy of SciencesUSA 85, 2444-2448 (1988)) or the BLAST algorithm of Altschul et al.(Nature Genetics 6, 119-129 (1994)) and compared with the sequenceentries which exist in databanks accessible to the public. Databanks fornucleotide sequences which are accessible to the public are, forexample, that of the European Molecular Biologies Laboratories (EMBL,Heidelberg, Germany) of that of the National Center for BiotechnologyInformation (NCBI, Bethesda, Md., USA).

The invention provides the new DNA sequence from C. glutamicum whichcontains the DNA section which codes for the tal gene, shown as SEQ IDNO 1 and SEQ ID NO 3. The amino acid sequence of the correspondingprotein has furthermore been derived from the present DNA sequence usingthe methods described above. The resulting amino acid sequence of thetal gene product is shown in SEQ ID NO 2 and SEQ ID NO 4.

A gene library produced in the manner described above can furthermore beinvestigated by hybridization with nucleotide probes of known sequence,such as, for example, the zwf gene (JP-A-09224661). The cloned DNA ofthe clones which show a positive reaction in the hybridization issequenced in turn to give on the one hand the known nucleotide sequenceof the probe employed and on the other hand the adjacent new DNAsequences.

Coding DNA sequences which result from SEQ ID NO 3 by the degeneracy ofthe genetic code are also a constituent of the invention. In the sameway, DNA sequences which hybridize with SEQ ID NO 3 or parts of or [sic]SEQ ID NO 3 are a constituent of the invention. Conservative amino acidexchanges, such as e.g. exchange of glycine for alanine or of asparticacid for glutamic acid in proteins, are furthermore known among expertsas “sense mutations” which do not lead to a fundamental change in theactivity of the protein, i.e. are of neutral function. It is furthermoreknown that changes on the N and/or C terminus of a protein cannotsubstantially impair or can even stabilize the function thereof.Information in this context can be found by the expert, inter alia, inBen-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), inO'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (ProteinSciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology6:1321-1325 (1988)) and in known textbooks of genetics and molecularbiology. Amino acid sequences which result in a corresponding mannerfrom SEQ ID NO 2 or SEQ ID NO 4 are also a constituent, of theinvention.

In the same way, DNA sequences which hybridize with or [sic] SEQ ID NO 3or parts of or [sic] SEQ ID NO 3 are a constituent of the invention.Finally, DNA sequences which are prepared by the polymerase chainreaction (PCR) using primers which result from SEQ ID NO 3 are aconstituent of the invention. Such oligonucleotides typically have alength of at least 15 nucleotides.

Instructions for identifying DNA sequences by means of hybridization canbe found by the expert, inter alia, in the handbook “The DIG SystemUsers Guide for Filter Hybridization” from Boehringer Mannheim GmbH(Mannheim, Germany, 1993) and in Liebl et al. (International Journal ofSystematic Bacteriology (1991) 41: 255-260). Instructions foramplification of DNA sequences with the aid of the polymerase chainreaction (PCR) can be found by the expert, inter alia, in the handbookby Gait: Oligonukleotide [sic] synthesis: a practical approach (IRLPress, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994).

The inventors have found that coryneform bacteria produce amino acids inan improved manner after over-expression of the tal gene.

To achieve an over-expression, the number of copies of the correspondinggenes can be increased, or the promoter and regulation region or theribosome binding site upstream of the structural gene can be mutated.Expression cassettes which are incorporated upstream of the structuralgene act in the same way. By inducible promoters, it is additionallypossible to increase the expression in the course of fermentativeL-amino acid production. The expression is likewise improved by measuresto prolong the life of the mRNA. Furthermore, the enzyme activity isalso increased by preventing the degradation of the enzyme protein. Thegenes or gene constructs can either be present in plasmids with avarying number of copies, or can be integrated and amplified in thechromosome. Alternatively, an over-expression of the genes in questioncan furthermore be achieved by changing the composition of the media andthe culture procedure.

Instructions in this context can be found by the expert, inter alia, inMartin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al.(Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6,428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), inEuropean Patent Specification EPS 0 472 869, in U.S. Pat. No. 4,601,893,in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheidet al. (Applied and Environmental Microbiology 60, 126-132 (1994)), inLaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), inPatent Application WO 96/15246, in Malumbres et al. (Gene 134, 15-24(1993)), in Japanese Laid-Open Specification JP-A-10-229891, in Jensenand Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inMakrides (Microbiological Reviews 60:512-538 (1996)) and in knowntextbooks of genetics and molecular biology.

By way of example, the tal gene according to the invention wasover-expressed with the aid of plasmids.

Suitable plasmids are those which are replicated in coryneform bacteria.Numerous known plasmid vectors, such as e.g. pZ1 (Menkel et al., Appliedand Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns etal., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74(1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Otherplasmid vectors, such as e.g. those based on pCG4 (U.S. Pat. No.4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66,119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in thesame manner.

Plasmid vectors which are furthermore suitable are also those with theaid of which the process of gene amplification by integration into thechromosome can be used, as has been described, for example, byReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)) for duplication or amplification of the hom-thrB operon. In thismethod, the complete gene is cloned in a plasmid vector which canreplicate in a host (typically E. coli), but not in C. glutamicum.Possible vectors are, for example, pSUP301 (Simon et al., Bio/Technology1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73(1994)), pGEM-T (Promega corporation [sic], Madison, Wis., USA),pCR2.1-TOPO (Shuman (1994). [sic] Journal of Biological Chemistry269:32678-84; U.S. Pat. No. 5,487,993), pCR® Blunt (Invitrogen,Groningen, Holland; Bernard et al., Journal of Molecular Biology, 234:534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41:337-342). Theplasmid vector which contains the gene to be amplified is thentransferred into the desired strain of C. glutamicum by conjugation ortransformation. The method of conjugation is described, for example, bySchäfer et al. (Applied and Environmental Microbiology 60, 756-759(1994)). Methods for transformation are described, for example, byThierbach et al. (Applied Microbiology and Biotechnology 29, 356-362(1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) andTauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). Afterhomologous recombination by means of a “cross over” event, the resultingstrain contains at least two copies of the gene in question.

An example of a plasmid vector with the aid of which the process ofamplification by integration can be carried out is pSUZ1, which is shownin FIG. 1. Plasmid pSUZ1 consists of the E. coli vector pBGS8 describedby Spratt et al. (Gene 41: 337-342 (1986)), into which the tal gene hasbeen incorporated.

In addition, it may be advantageous for the production of amino acids toamplify or over-express one or more enzymes of the particularbiosynthesis pathway, of glycolysis, of anaplerosis, of the pentosephosphate pathway or of amino acid export, in addition to the tal gene.

Thus, for example, for the preparation of L-amino acids, in particularL-lysine, one or more genes chosen from the group consisting of

-   -   the dapA gene which codes for dihydrodipicolinate synthase (EP-B        0 197 335),    -   the lysC gene which codes for a feed back resistant aspartate        kinase (Kalinowski et al. (1990), Molecular and General Genetics        224: 317-324),    -   the gap gene which codes for glycerolaldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086),    -   the pyc gene which codes for pyruvate carboxylase (DE-A-198 31        609),    -   the mqo gene which codes for malate:quinone oxidoreductase        (Molenaar et al., European Journal of Biochemistry 254, 395-403        (1998)),    -   the tkt gene which codes for transketolase (accession number        AB023377 of the databank of European Molecular Biologies        Laboratories (EMBL, Heidelberg, Germany)),    -   the gnd gene which codes for 6-phosphogluconate dehydrogenase        (JP-A-9-224662),    -   the zwf gene which codes for glucose 6-phosphate dehydrogenase        (JP-A-9-224661),    -   the lysE gene which codes for lysine export (DE-A-195 48 222),    -   the zwa1 gene (DE 199 59 328.0; DSM 13115),    -   the eno gene which codes for enolase (DE: 19947791.4),    -   the devB gene,    -   the opcA gene (DSM 13264)        can be amplified, preferably over-expressed, at the same time.

Thus, for example, for the preparation of L-threonine, one or more geneschosen from the group consisting of

-   -   at the same time the hom gene which codes for homoserine        dehydrogenase (Peoples et al., Molecular Microbiology 2, 63-72        (1988)) or the hom^(dr) allele which codes for a “feed back        resistant” homoserine dehydrogenase (Archer et al., Gene 107,        53-59 (1991),    -   the gap gene which codes for glycerolaldehyde 3-phosphate        dehydrogenase (Eikmanns (1992), Journal of Bacteriology        174:6076-6086),    -   the pyc gene which codes for pyruvate carboxylase (DE-A-198 31        609),    -   the mqo gene which codes for malate:quinone oxidoreductase        (Molenaar et al., European Journal of Biochemistry 254, 395-403        (1998)),    -   the tkt gene which codes for transketolase (accession number        AB023377 of the databank of European Molecular Biologies        Laboratories (EMBL, Heidelberg, Germany)),    -   the gnd gene which codes for 6-phosphogluconate dehydrogenase        (JP-A-9-224662),    -   the zwf gene which codes for glucose 6-phosphate dehydrogenase        (JP-A-9-224661),    -   the thrE gene which codes for threonine export (DE 199 41 478.5;        DSM 12840),    -   the zwa1 gene (DE 199 59 328.0; DSM 13115),    -   the eno gene which codes for enolase (DE: 19947791.4),    -   the devB gene,    -   the opcA gene (DSM 13264)        can be amplified, preferably over-expressed, at the same time .        . . [sic]

It may furthermore be advantageous for the production of amino acids toattenuate

-   -   the pck gene which codes for phosphoenol pyruvate carboxykinase        (DE 199 50 409.1 DSM 13047) and/or    -   the pgi gene which codes for glucose 6-phosphate isomerase (U.S.        Ser. No. 09/396,478, DSM 12969), or    -   the poxB gene which codes for pyruvate oxidase (DE 199 51 975.7;        DSM 13114), or    -   the zwa2 gene (DE: 199 59 327.2; DSM 13113)        at the same time, in addition to the amplification of the tal        gene.

In addition to over-expression of the tal gene it may furthermore beadvantageous for the production of amino acids to eliminate undesirableside reactions (Nakayama: “Breeding of Amino Acid ProducingMicro-organisms”, in: Overproduction of Microbial Products, Krumphanzl,Sikyta, Vanek (eds.), Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention can be culturedcontinuously or discontinuously in the batch process (batch culture) orin the fed batch (feed process) or repeated fed batch process(repetitive feed process) for the purpose of production of L-aminoacids. A summary of known culture methods are [sic] described in thetextbook by Chmiel (Bioprozeβtechnik 1. Einführung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren and periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of theparticular strains in a suitable manner. Descriptions of culture mediafor various microorganisms are contained in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981). Sugars and carbohydrates,such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses,starch and cellulose, oils and fats, such as e.g. soya oil, sunfloweroil, groundnut oil and coconut fat, fatty acids, such as e.g. palmiticacid, stearic acid and linoleic acid, alcohols, Such as e.g. glyceroland ethanol, and organic acids, such as e.g. acetic acid, can be used asthe source of carbon. These substances can be used individually or as amixture. Organic nitrogen-containing compounds, such as peptones, yeastextract, meat extract, malt extract, corn steep liquor, soya bean flourand urea, or inorganic compounds, such as ammonium sulphate, ammoniumchloride, ammonium phosphate, ammonium carbonate and ammonium nitrate,can be used as the source of nitrogen. The sources of nitrogen can beused individually or as a mixture. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen phosphate or the correspondingsodium-containing salts can be used as the source of phosphorus. Theculture medium must furthermore comprise salts of metals, such as e.g.magnesium sulfate or iron sulfate, which are necessary for growth.Finally, essential growth substances, such as amino acids and vitamins,can be employed in addition to the abovementioned substances. Suitableprecursors can moreover be added to the culture medium. The startingsubstances mentioned can be added to the culture in the form of a singlebatch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammoniaor aqueous ammonia, or acid compounds, such as phosphoric acid orsulfuric acid, can be employed in a suitable manner to control the pH ofthe culture. Antifoams, such as e.g. fatty acid polyglycol esters, canbe employed to control the development of foam. Suitable substanceshaving a selective action, such as e.g. antibiotics, can be added to themedium to maintain the stability of plasmids. To maintain aerobicconditions, oxygen or oxygen-containing gas mixtures, such as e.g. air,are introduced into the culture. The temperature of the culture isusually 20° C. to 45° C., and preferably 25° C. to 40° C. Culturing iscontinued until a maximum of L-amino acid has formed. This target isusually reached within 10 hours to 160 hours.

The analysis of L-amino acids can be carried out by anion exchangechromatography with subsequent ninhydrin derivatization, as described bySpackman et al. (Analytical Chemistry, 30, (1958), 1190).

The following microorganism has been deposited at the Deutsche Sammlungfür Mikrorganismen and Zellkulturen (DSMZ=German Collection ofMicroorganisms and Cell Cultures, Braunschweig, Germany) in accordancewith the Budapest Treaty:

-   -   Escherichia coli JM109/pSUZ1 as DSM 13263.

SEQ ID NO 1 also contains the new devB gene. The process according tothe invention is used for fermentative preparation of amino acids.

The following figures are attached:

FIG. 1: Map of the plasmid pSUZ1

The abbreviations and designations used have the following meaning.

lacZ: segments of lacZα gene fragmentkan r: kanamycin resistancetal: transaldolase geneori: origin of replication of plasmid pBGS8BclI: cleavage site of restriction enzyme BclIEcoRI: cleavage site of restriction enzyme EcoRIHindIII: cleavage site of restriction enzyme HindIIIPstI: cleavage site of restriction enzyme PstISacI: cleavage site of restriction enzyme SacI

EXAMPLES

The following examples will further illustrate this invention. Themolecular biology techniques, e.g. plasmid DNA isolation, restrictionenzyme treatment, ligations, standard transformations of Escherichiacoli etc. used are, (unless stated otherwise), described by Sambrook etal., (Molecular Cloning. A Laboratory Manual (1989) Cold Spring HarbourLaboratories, USA).

Example 1 Preparation of a Genomic Cosmid Gene Library fromCorynebacterium glutamicum ATCC 13032

Chromosomal DNA from Corynebacterium glutamicum ATCC 13032 was isolatedas described by Tauch et al. (1995, Plasmid 33:168-179) and partlycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Molecular Biochemicals, Mannheim, Germany, Product DescriptionSAP, Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl etal. (1987) Proceedings of the National Academy of Sciences USA84:2160-2164), obtained from Stratagene (La Jolla, USA, ProductDescription SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleavedwith the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,Product Description XbaI, Code no. 27-0948-02) and likewisedephosphorylated with shrimp alkaline phosphatase. The cosmid DNA wasthen cleaved with the restriction enzyme BamHI (Amersham Pharmacia,Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). Thecosmid DNA treated in this manner was mixed with the treated ATCC13032DNA and the batch was treated with T4 DNA ligase (Amersham Pharmacia,Freiburg, Germany, Product Description T4-DNA-Ligase, Code no.27-0870-04). The ligation mixture was then packed in phages with the aidof Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, ProductDescription Gigapack II XL Packing Extract, Code no. 200217). Forinfection of the E. coli strain NM554 (Raleigh et al. 1988, Nucleic AcidResearch 16:1563-1575) the cells were taken up in 10 mM MgSO₄ and mixedwith an aliquot of the phage suspension. The infection and titering ofthe cosmid library were carried out as described by Sambrook et al.(1989, Molecular Cloning: A laboratory Manual, Cold Spring Harbor), thecells being plated out on LB agar (Lennox, 1955, Virology, 1:190) with100 μg/ml ampicillin. After incubation overnight at 37° C., recombinantindividual clones were selected.

Example 2 Isolation and Sequencing of the tal Gene

The cosmid DNA of an individual colony was isolated with the QiaprepSpin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) inaccordance with the manufacturer's instructions and partly cleaved withthe restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,Product Description Sau3AI, Product No. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (Roche MolecularBiochemicals, Mannheim, Germany, Product Description SAP, Product No.1758250). After separation by gel electrophoresis, the cosmid fragmentsin the size range of 1500 to 2000 bp were isolated with the QiaExII GelExtraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA ofthe sequencing vector pZero-1, obtained from Invitrogen (Groningen,Holland, Product Description Zero Background Cloning Kit, Product No.K2500-01) was cleaved with the restriction enzyme BamHI (AmershamPharmacia, Freiburg, Germany, Product Description BamHI, Product No.27-0868-04). The ligation of the cosmid fragments in the sequencingvector pZero-1 was carried out as described by Sambrook et al. (1989,Molecular Cloning: A laboratory Manual, Cold Spring Harbor), the DNAmixture being incubated overnight with T4 ligase (Pharmacia Biotech,Freiburg, Germany). This ligation mixture was then electroporated (Tauchet al. 1994, FEMS Microbiol Letters, 123:343-7) into the E. coli strainDH5αMCR (Grant, 1990, Proceedings of the National Academy of SciencesU.S.A., 87:4645-4649) and plated out on LB agar (Lennox, 1955, Virology,1:190) with 50 μg/ml zeocin. The plasmid preparation of the recombinantclones was carried out with Biorobot 9600 (Product No. 900200, Qiagen,Hilden, Germany). The sequencing was carried out by the dideoxychain-stopping method of Sanger et al. (1977, Proceedings of theNational Academy of Sciences U.S.A., 74:5463-5467) with modificationsaccording to Zimmermann et al. (1990, Nucleic Acids Research, 18:1067).The “RR dRhodamin Terminator Cycle Sequencing Kit” from PE AppliedBiosystems (Product No. 403044, Weiterstadt, Germany) was used. Theseparation by gel electrophoresis and analysis of the sequencingreaction were carried out in a “Rotiphoresis NFAcrylamide/Bisacrylamide” Gel (29:1) (Product No. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogram package (1986, Nucleic Acids Research, 14:217-231) version 97-0:The individual sequences of the pZero1 derivatives were assembled to acontinuous contig. The computer-assisted coding region analysis [sic]were prepared with the XNIP program (Staden, 1986, Nucleic AcidsResearch, 14:217-231). Further analyses were carried out with the “BLASTsearch program” (Altschul et al., 1997, Nucleic Acids Research,25:3389-3402), against the non-redundant databank of the “NationalCenter for Biotechnology Information” (NCBI, Bethesda, Md., USA). Thenucleotide sequence obtained is shown in SEQ ID NO 1 and SEQ ID NO 3.

Example 3 Cloning of the tal Gene

PCR was used to amplify DNA fragments containing the entire tal gene ofC. glutamicum 13032 and flanking upstream and downstream regions. PCRreactions were carried out using oligonucleotide primers designed fromthe sequence as determined in examples 1 and 2. Genomic DNA was isolatedfrom Corynebacterium glutamicum ATCC13032 according to Heery and Dunican(Applied and Environmental Microbiology 59: 791-799 (1993)) and used astemplate. The tal primers used were:

fwd. primer: 5′ GGT ACA AAG GGT CTT AAG 3′Crev. primer: 5′ GAT TTC ATG TCG CCG TTA 3′

PCR Parameters were as follows:

-   -   35 cycles    -   95° C. for 0.3 minutes    -   94° C. for 1 minute    -   47° C. for 1 minute    -   72° C. for 45 seconds    -   2.0 mM MgCl2    -   approximately 150-200 ng DNA template.

The PCR product obtained was cloned into the commercially availablepGEM-T vector purchased from Promega Corp. (pGEM-T Easy Vector System 1,cat. no. A1360, Promega UK, Southampton, UK) using strain E. coli JM109(Yanisch-Perron et al., Gene, 33: 103-119 (1985)) as a host. The entiretal gene was subsequently isolated from the pGEM T-vector on an Eco RIfragment and cloned into the lacZα EcoRI site of the E. coli vectorpBGS8 (Spratt et al., Gene 41(2-3): 337-342 (1986)). The restrictionenzymes used were obtained from Boehringer Mannheim UK Ltd. (Bell Lane,Lewes East Sussex BN7 1LG, UK) and used according to manufacturer'sinstructions. E. coli JMI09 was then transformed with this ligationmixture and electrotransformants were selected on Luria agarsupplemented with isopropyl-thiogalactopyranoside (IPTG),5-bromo-4-chloro-3-indolyl-galactopyranoside (XGAL) and kanamycin atconcentrations of 1 mM, 0.02% and 50 mg/l respectively. Plates wereincubated for twelve hours at 37° C. Plasmid DNA was isolated from onetransformant, characterised by restriction enzyme analysis using Eco RI.This new construct was designated pSUZ 1.

1-7. (canceled)
 8. A process for the preparation of L-amino acids, whichcomprises carrying out the following steps: a) fermentation of thebacteria which produce the desired L-amino acid, in which at least thetal gene and optionally one or more of the genes tkt gene, zwt gene,devB gene or opcA gene are amplified at the same time, b) concentrationof the desired product in the medium or in the cells of the bacteria andc) isolation of the desired L-amino acid.
 9. A process as claimed inclaim 8, wherein bacteria in which further genes of the biosynthesispathway of the desired L-amino acid are additionally amplified areemployed.
 10. A process as claimed in claim 8, wherein bacteria in whichthe metabolic pathways which reduce the formation of the desired L-aminoacid are at least partly eliminated are employed. 11-16. (canceled)